Investigational New Drugs

, Volume 34, Issue 2, pp 159–167 | Cite as

The novel pyrrolo-1,5-benzoxazepine, PBOX-15, synergistically enhances the apoptotic efficacy of imatinib in gastrointestinal stromal tumours; suggested mechanism of action of PBOX-15

  • Paula Kinsella
  • Lisa M. Greene
  • Sandra A. Bright
  • Jade K. Pollock
  • Stefania Butini
  • Giuseppe Campiani
  • Sebastian Bauer
  • D. Clive Williams
  • Daniela M. Zisterer
PRECLINICAL STUDIES

Summary

The C-KIT receptor tyrosine kinase is constitutively activated in the majority of gastrointestinal stromal tumours (GIST). Imatinib (IM) a selective inhibitor of C-KIT, is indicated for the treatment of KIT-positive unresectable and/or metastatic GIST, and has tripled the survival time of patients with metastatic GIST. However, the majority of patients develop IM-resistance and progress. Although IM elicits strong antiproliferative effects, it fails to induce sufficient levels of apoptosis; acquired IM-resistance and disease recurrence remain an issue, a more effective drug treatment is greatly needed. We examined the effect of a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-15 in combination with IM on GIST cells. PBOX-15 decreased viability and in combination with IM synergistically enhanced apoptosis in both IM-sensitive and IM-resistant GIST cells, decreased the anti-apoptotic protein Mcl-1, and enhanced activation of pro-caspase-3 and PARP cleavage. The combination treatment also led to an enhanced inhibition of C-KIT-phosphorylation and inactivation of C-KIT-dependent signalling in comparison to either drug alone; CDC37, a key regulator of C-KIT in GIST was also dramatically decreased. Furthermore, PBOX-15 reduced CKII expression, an enzyme which regulates the expression of CDC37. In conclusion, our findings indicate the potential of PBOX-15 to improve the apoptotic response of IM in GIST cells and provide a more effective treatment option for GIST patients.

Keywords

Gastrointestinal Imatinib Microtubules CKIT CDC37 CKII 

Introduction

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumour of the gastrointestinal tract and are characterised by activating mutations, usually in exon-9 or −11, of the stem cell receptor tyrosine kinase (C-KIT) (85 %) or the platelet-derived growth factor receptor A kinase (PDGFRA) (7 %) [1, 2]. Targeted therapy with the tyrosine kinase inhibitor imatinib mesylate (IM; Glivec®, Novartis Pharmaceuticals), which inhibits KIT and PDGFRA activity is a highly effective treatment for metastatic GIST. Prior to the use of IM, recurrent or metastatic GIST was uniformly fatal. IM was originally developed as an inhibitor of the ATP binding site of the breakpoint cluster region-abelson (BCR-ABL) fusion onco-protein, which plays a central role in the oncogenesis of chronic myelogenous leukaemia (CML) [3]. IM also targets the ATP binding site of KIT, causing inhibition of autophosphorylation, and disruption of the downstream signalling pathways involved in cellular proliferation and survival such as mitogen-activated protein (MAP) kinase and AKT signalling pathways [4].

Although >85 % of patients with metastatic GIST benefit from IM therapy, complete responses are rare and the majority of patients develop resistance to IM during the course of treatment. It is critical to identify novel strategies or new agents that induce GIST cell apoptosis either as single agents or in combination with IM.

Recently we identified a series of novel MTAs, the PBOX compounds, which behave as tubulin depolymerisers and possess the ability to potently induce apoptosis in several cancerous cell lines [5, 6, 7]. The PBOXs also induce apoptosis in ex vivo chronic lymphocytic leukaemia [8] and CML patient samples including those that are resistant to IM [9]. We have shown that PBOXs impair the growth of tumours in vivo in breast cancer and CML mouse tumour models. As GIST and CML are both cancers characterised by a unique oncogenic dependency on a tyrosine kinase we sought to evaluate the therapeutic potential of PBOX-15 in GIST. Collectively these results suggest the PBOXs are a promising group of potential anti-cancer therapeutics. We decided to examine the potential of combining IM with PBOX-15 as a novel treatment option for GIST. We hoped to decrease cell viability and increase the apoptosis in GIST cells by targeting both C-KIT with IM and microtubules with PBOX-15.

Materials and methods

Cell culture

GIST-T1 was developed by Takahiro Taguchi (Kochi University, Kochi, Japan) an IM-sensitive metastatic GIST and GIST-T1-Juke is an IM-resistant subline that has a secondary KIT-resistance mutation D816E [10]. GIST-T1 cells cultured in DMEM/F12 + GlutaMAX medium supplemented with 10 % (v/v) foetal bovine serum (FBS) and 1 % (v/v) penicillin/streptomycin. GIST-T1-Juke cells grown in RPMI 1640 + GlutaMAX medium supplemented with 15 % (v/v) (FBS) and 1 % (v/v) penicillin/streptomycin. Cells were incubated in a humidified environment at 95 % O2 and 5 % CO2, passaged twice a week.

Reagents

Pyrrolo-1,5-benzoxazepine-15 (PBOX-15) was synthesised as described previously [11] dissolved in ethanol and stored at -20 °C. Imatinib (Novartis) was reconstituted in DMSO (10 mM stock) and stored at -20 °C. Media sourced from Biosciences, and FBS obtained from Gemini Bioproducts. The BCA reagents from Pierce (Illinois, U.S.), the polyvinylidene difluoride membranes from Millipore (Cork, Ireland), protease inhibitors from Roche (Clare, Ireland). Chemicals from Sigma-Aldrich (Sigma Aldrich, St Louis, MO, USA), cell culture materials from Greiner Bio-One GmbH (Stonehouse, U.K.).

AlamarBlue monolayer viability assay

Cells were seeded at densities varying from 4 000–5 000 cells/well, 96-well plate, 200 μL medium, left overnight to attach, treated with a range of concentrations of PBOX-15 and IM for 24 or 48 h. AlamarBlue (final concentration 10 % (v/v)) added to each well and left to incubate in the dark at 37 °C for 5 h. Fluorescence was measured using a SpectraMax Gemini plate reader (Molecular Devices, Sunnyvale, CA) at excitation and emission wavelengths of 544 nm and 590 nm. The mean of each triplicate was calculated. Vehicle treated wells were set at 100 % viability and treated wells were calculated as a percentage of the vehicle control. Dose response curves were plotted and IC50 values obtained using Prism GraphPad 4.

Flow cytometry

Following treatment, cells were harvested and fixed in 70 % ethanol/PBS. Fixed samples were stored at -20 °C until required. 4 mLs of PBS was added to each sample, ethanol was removed by centrifugation, pellets were incubated in 400 μL FACS flow sheath fluid supplemented with 10 mg/mL RNase A (Sigma Aldrich,

St Louis, MO, USA) and 100 mg/mL propidium iodide (PI) (Sigma Aldrich, St Louis, MO, USA). Cells were incubated in the dark at 37 °C for 30 mins. Analysis was performed on a FACScalibur Fluorescence Associated Cell Sorter (FACS) (Becton–Dickinson, San Jose, CA, USA) using Cell Quest and Quanti-Quest software. Samples were gated using a vehicle control to eliminate debris and cell aggregates from analysis. 10,000 cells from each sample were counted and results visualised on histrograms.

Analysis of drug interactions

Using the software programme Calcusyn, median dose analysis was undertaken to study interactions between different drugs. This method is based on the drug effect equation of Chao and Talalay [12] providing quantitative determination for synergism. The lower the combination index value (CI), the greater the degree of synergism (CI < 1), additive effect (CI = 1) and antagonism (CI > 1). A CI value of 0.1–0.3 indicates strong synergism, 0.3–0.7 indicates synergism, 0.7–0.85 indicates moderate synergism and 0.85–0.9 indicates slight synergism.

Statistics

Statistical analysis of experimental data was performed using the computer program Prism Graph Pad 4. Results were presented as mean ± S.E.M. For comparison of two groups, values were determined using a Student’s paired t test. A value of P < 0.05 was deemed to be significant.

Western blotting

Cells were harvested in Ripa buffer (R0278, Sigma-Aldrich); DTT (to a final concentration of 50 mM), 0.313 M Tris HCL pH 6.8, 10 % SDS and 0.25 % bromophenol blue, 50 % glycerol were added to each sample followed by boiling for 4 mins at 95 °C. Equal quantities of protein (as determined by a BCA assay) were separated on polyacrylamide gels followed by transfer to PVDF membranes. Membranes were blocked in 5 % blocking serum (Bio-Rad) in PBS-tween (0.1 %) for 1 h. Membranes were incubated in the relevant primary antibodies overnight, washed and incubated in secondary antibody for 1 h and washed again. Enhanced chemiluminescence (Millipore, Cork, Ireland) was used for detection of protein expression. Western blot analysis was performed using antibodies directed against PARP (AM30, Millipore), Pro-Caspase-3 (Merck Biosciences), Mcl-1 (Am50, Calbiochem), P-C-KIT (Tyr719, Cell Signalling (CS), C-KIT (Ab81, CS), P-Akt (S473, CS), Akt (C67E7, CS), P-PTEN (S380, CS), P-CDC37 (Ser13, CS), CDC37 (D28H7, CS), HSP90 (610,418, BD Biosciences), CKIIα (A300-196 A-T, Bethyl), (with incubation with a horseradish peroxidase-conjugated anti-mouse, anti-rabbit antibody (Promega, Madison, WI, USA)). Blots were probed with β-actin to confirm equal loading. 48 h β-actin is shown and is representative of equal loading of all blots.

Results

Imatinib and PBOX-15 reduce the viability of GIST cells in an AlmarBlue monolayer viability assay

An IM-sensitive (GIST-T1) and an IM-resistant (GIST-T1-Juke) cell line were treated with a range of concentrations of IM or PBOX-15 for 48 h, cell viability was measured using the AlamarBlue monolayer viability assay. With IM treatments the GIST-T1 gave an IC50 value of 0.07 ± 0.005 μM (Fig. 1a) while as expected the IM-resistant cell line GIST-T1-Juke gave a much higher IC50 of 2.7 ± 0.5 μM, values are similar to previous reports [13, 14, 15]. GIST-T1 gave an IC50 value of 0.7 ± 0.1 μM with PBOX-15. An IC50 value was not achieved with GIST-T1-Juke with PBOX-15 over 48 h, although an IC20 value of 3.1 ± 0.7 μM (Fig. 1b) was obtained. We tested a higher concentration of PBOX-15 (16 μM) in both cell lines, however, no increase in toxicity was seen.
Fig. 1

Imatinib and PBOX-15 reduce the viability of GIST cells. GIST-T1 and GIST-T1-Juke were treated with a range of concentration of either (a) imatinib (0.01 μM to 5 μM) or (b) PBOX-15 (0.25 μM to 4 μM) for 48 h and cell viability was assessed with an AlamarBlue viability assay. Cells treated with vehicle (Veh) were set to 100 % cell survival. Data represent the mean +/− S.E.M. of three experiments. IC50 and IC20 values were obtained using Prism Graphpad 4

PBOX-15 synergistically enhances the apoptotic effect of imatinib in both sensitive and resistant GIST cells via flow cytometric analysis

Flow cytometric analysis of PI stained cells was used to assess the effects of IM and PBOX-15, on cell cycle progression and induction of apoptosis. The apoptotic population was quantified as the percentage of cells in the sub G0/G1 fraction. Cells were treated with a range of concentrations of PBOX-15 with IM (based on the viability results) for 48 h. We used a software package, Calcusyn, to analyse the synergy of the apoptosis levels. The percentage of apoptotic cells increased with increasing concentrations of both drugs. In GIST-T1 cells, IM (0.16 μM) and PBOX-15 (0.6 μM) gave the most apoptosis (52.2 %) and a strong synergistic effect with Calcusyn, CI value of 0.161, indicating strong synergism between the two drugs (Table 1). In GIST-T1-Juke, IM (16 μM) and PBOX-15 (3.2 μM) gave the most apoptosis (34.8 %) a strong synergistic effect with Calcusyn, CI value of 0.208, indicating strong synergism between the two drugs in these resistant GIST cells (Table 2).
Table 1

PBOX-15/imatinib combination synergistically enhance apoptosis in GIST-T1 (imatinib-sensitive GIST cells)

a

 

Imatinib (μM)

% Sub G1/G0 Cells

0.01

0.49

0.04

1.4

0.16

9.51

PBOX-15 (μM)

0.15

0.97

0.6

18.35

2.4

6.05

b

Imatinib (μM)

PBOX-15 (μM)

% Sub G1/G0 Cells

Fa

CI

0.01

0.15

0.9

0.008

3.188

0.01

0.6

18

0.151

0.191

0.01

2.4

8.3

0.096

1.364

0.04

0.15

2.9

0.031

1.007

0.04

0.6

36.6

0.337

0.073

0.04

2.4

38

0.358

0.175

0.16

0.15

13.5

0.093

0.783

*0.16

0.6

52.2

0.346

0.161

0.16

2.4

42.4

0.33

0.304

Using the software package Calcusyn, the percentage of cells undergoing apoptosis (sub-G0/G1 peak) was measured by flow cytometric analysis for nine different concentration combinations of imatinib and PBOX-15 as indicated. A CI value of >1 showed antagonism, =1 additive and <1 indicated synergism. n = 3

Fa fraction affected

CI Combination Index

*The combination we chose to further examine as it gave the highest amount of apoptosis at 48 h

Table 2

PBOX-15/imatinib combination synergistically enhance apoptosis in GIST-T1-Juke (imatinib-resistant GIST cells)

a

 

Imatinib (μM)

% Sub G1/G0 Cells

4

3.4

8

4.6

16

5.7

PBOX-15 (μM)

1.6

12.3

3.2

15.3

6.4

24.5

b

Imatinib (μM)

PBOX-15 (μM)

% Sub G1/G0 Cells

Fa

CI

4

1.6

12

0.12

0.980

4

3.2

18.9

0.19

0.770

4

6.4

22.7

0.23

1.017

8

1.6

19.8

0.2

0.377

8

3.2

20.8

0.21

0.644

8

6.4

24.7

0.25

0.860

16

1.6

22.5

0.22

0.340

*16

3.2

34.8

0.35

0.208

16

6.4

32.3

0.32

0.498

Using the software package Calcusyn, the percentage of cells undergoing apoptosis (sub-G0/G1 peak) was measured by flow cytometric analysis for nine different concentration combinations of imatinib and PBOX-15 as indicated. A CI value of >1 showed antagonism, =1 additive and <1 indicated synergism. n = 3

Fa fraction affected

CI Combination Index

*The combination we chose to further examine through FACS and western blotting as it gave the highest amount of apoptosis at 48 h

The effect of IM and PBOX-15 on cell cycle progression in GIST-T1 cells was also established at 24 h. IM (0.16 μM) induced G0/G1 arrest, PBOX-15 (0.6 μM) induced a marked G2/M arrest (67 %), consistent with previous reports [16] demonstrating PBOX-15 targets the microtubules. Minimal apoptosis was induced by either drug alone or in combination at 24 h (Fig. 2a). At 48 h, IM alone further induced the G0/G1 arrest (80 %) and exhibited a low percentage of apoptosis (9 %). PBOX-15 resulted in a decrease of cells in G2/M (19 %) and an increase in cells undergoing apoptosis (18 %) (Fig. 2a). The combination significantly enhanced the percentage of apoptotic cells (52 %) when compared to either drug alone (Fig. 2a). Similar results were obtained at 48 h with the GIST-T1-Juke cells, the combination elicited 32 % apoptosis when compared to 5 % and 16 % when treated with either IM or PBOX-15 alone.
Fig. 2

PBOX-15 induced G2/M arrest precedes apoptosis in GIST cells. (a) Cell cycle phases of GIST-T1 including Sub-G0/G1, G0/G1, S and G2M at 24 h and 48 h treated with imatinib (0.16 μM), PBOX-15 (0.6 μM) or a combination. Cell cycle phases of GIST-T1-Juke at 48 h treated with imatinib (16 μM), PBOX-15 (3.2 μM) or a combination. Data represents the mean ± S.E.M. of three experiments. Statistical analysis was performed using a Student’s t-test. P values correspond to sub-G0/G1. (b) Apoptosis was confirmed by western blot analysis of pro-caspase-3 and of PARP cleavage. Cells were treated with vehicle (Veh), imatinib (0.16 μM), PBOX-15 (0.6 μM) or combination in GIST-T1 cells for 48 h or with vehicle (Veh), imatinib (16 μM), PBOX-15 (3.2 μM) or combination in GIST-T1-Juke cells β-actin expression was measured as a loading control. Results are representative of three separate experiments

Western blot analysis of pro-caspase-3 and of poly (ADP) ribose polymerase (PARP) cleavage in GIST cells confirmed the sub G0/G1 peaks observed by flow cytometric analysis were representative of an apoptotic cell population. Consistent with results from flow cytometry, the combination caused the disappearance of the 32 kDa band of pro-caspase-3 indicating its activation (Fig. 2b). Enhanced PARP cleavage was observed with the combination in both GIST-T1 and Juke cells in comparison to either agent alone.

PBOX-15 treatment is accompanied by an enhanced downregulation of anti-apoptotic mcl-1 in GIST-T1 and GIST-T1-juke cells

To further confirm the apoptotic effect of IM and PBOX-15 in GIST-T1 cells, we examined the expression of an anti-apoptotic protein Mcl-1 at 24 and 48 h in GIST-T1 cells and at 48 h in GIST-T1-Juke cells. IM slightly decreased anti-apoptotic Mcl-1 at 48 h in GIST-T1 cells. A strong decrease in Mcl-1 was seen following PBOX-15 treatment over time in GIST-T1 cells with undetectable levels at 48 h, GIST-T1-Juke cells also had no expression of Mcl-1 at 48 h due to treatment with PBOX-15 (Fig. 3).
Fig. 3

Imatinib/PBOX-15 combination downregulates Mcl-1 in GIST-T1 and GIST-T1-Juke cells. Cells were treated with vehicle (Veh), imatinib (0.16 μM), PBOX-15 (0.6 μM) or combination for 24 and 48 h in GIST-T1 cells and imatinib (16 μM), PBOX-15 (3.2 μM) or combination for 48 h and cell lysates were prepared for western blot analysis. β-actin expression was measured as a loading control. Results are representative of three separate experiments

PBOX-15/imatinib combination inhibits KIT and KIT-Dependent signalling pathways in both imatinib-sensitive and imatinib-resistant GIST cells

To elucidate the mechanism of PBOX-15 induced sensitisation to apoptosis, we investigated the effects of PBOX-15 on KIT and KIT-dependent signalling pathways, alone and in combination with IM. Both IM and PBOX-15 suppressed C-KIT phosphorylation and total C-KIT expression in IM-sensitive GIST-T1 cells; the combination treatment gave enhanced inhibition of P-C-KIT at 24 and 48 h. This result was paralleled by a substantial inhibition of the downstream PI3K/Akt pathway as monitored by the suppression of both P-Akt at 24 h and total Akt at 48 h.

In the IM-resistant GIST-T1-Juke cells at 48 h IM increased total C-KIT and elicited a minimal effect on P-C-KIT, whereas PBOX-15 strongly decreased both total and phospho-C-KIT. The combination suppressed P-C-KIT compared to the IM treatment and this was paralleled by a downregulation in P-Akt and total Akt. P-PTEN, a negative regulator of the pathway was not affected by either drug in GIST-T1 or GIST-T1-Juke (Fig. 4).
Fig. 4

PBOX-15/imatinib combination inhibits C-KIT and C-KIT-dependent signalling pathways. Cells were treated with vehicle (Veh), imatinib (0.16 μM), PBOX-15 (0.6 μM) or combination in GIST-T1 cells for 24 or 48 h or with vehicle (Veh), imatinib (16 μM), PBOX-15 (3.2 μM) or combination in GIST-T1-Juke cells for 48 h. Cells were probed for total C-KIT (C-KIT), phosphorylated C-KIT (P-C-KIT), total Akt (Akt), phosphorylated Akt (P-Akt) or phosphorylated PTEN (P-PTEN). β-actin expression was measured as a loading control. Results are representative of three separate experiments

Imatinib and PBOX-15 supresses CDC37 in both imatinib sensitive and resistant GIST

The HSP90 chaperone has previously been shown to be required for folding, localisation and stabilisation of C-KIT in GIST [13]. CDC37 is a crucial HSP90-cofactor for KIT oncogenic expression in GIST cells [17]. We examined the effect of the drugs on CDC37 and HSP90 in GIST-T1 cells. Both IM and PBOX-15 decreased total CDC37 and P-CDC37, with the strongest effect seen with the combination in GIST-T1 (Fig. 5). A strong decrease in HSP90 in GIST-T1 was seen with IM and the combination possibly due to the effect of IM alone, as PBOX-15 did not appear to have any effect on HSP90, however in the GIST-T1-Juke cells HSP90 appears to be decreased by PBOX-15 (Fig. 5). In addition in GIST-T1 we looked at CKIIα expression, as this protein is involved in a positive feedback loop with CDC37. PBOX-15 alone decreased CKIIα with an enhanced effect observed with the combination in both cell lines (Fig. 5).
Fig. 5

PBOX-15/imatinib combination suppresses CDC37, the HSP90-cofactor for KIT oncogenic expression in GIST cells. Cells were treated with vehicle (Veh), imatinib (0.16 μM), PBOX-15 (0.6 μM) or combination in GIST-T1 cells or with vehicle (Veh), imatinib (16 μM), PBOX-15 (3.2 μM) or combination in GIST-T1-Juke cells for 48 h. Cells were probed for total CDC37, phosphorylated CDC37 (P-CDC37), HSP90 or CKIIα. β-actin expression was measured as a loading control. Results are representative of three separate experiments

Discussion

There is an urgent need for apoptosis inducing agents that can be administered in combination with IM. Studies have shown the use of a MTA in combination with IM to be very promising [18, 19]. We demonstrate for the first time the potential benefit of targeting KIT with IM while inhibiting the assembly of tubulin with the MTA, PBOX-15. We have shown IM combined with PBOX-15, synergistically enhances apoptosis in both IM-sensitive and IM-resistant GIST cells: 52 % increase in apoptosis in IM-sensitive cells and 34 % apoptosis in IM-resistant GIST cells. IM has previously been reported to induce cell cycle arrest at G0/G1 in GIST cells [20] whilst PBOX compounds induce G2M arrest [16], this correlates with this study, the targeting of the cell cycle at these two phases, may have resulted in the synergistic apoptotic effect seen. Treatment with the combination of IM and PBOX-15 resulted in enhanced activation of pro-caspase-3 and PARP cleavage in comparison to either agent alone which correlates with the flow cytometric data.

To further confirm the enhanced apoptotic effect of PBOX-15 and IM in our GIST cells we examined the expression of an anti-apoptotic protein Mcl-1 following treatment. Mcl-1 has been purported to be a critical regulator of apoptosis induced by MTAs [16, 21]. As PBOX-15 is a MTA we postulated that it may affect expression of Mcl-1 in GIST cells. Mcl-1 was strongly down-regulated by PBOX-15 in both IM-sensitive and –resistant GIST cells, consistent with previous PBOX studies [5, 16].

As GIST is highly dependent on the PI3K-Akt pathway for survival, in particular C-KIT expression, we examined the effect of the drug treatments on this pathway. PBOX-15 combined with IM had a much stronger inhibitory effect than IM alone on P-C-KIT in both IM-resistant and IM-sensitive GIST cell lines, this correlated with a strong decrease in Akt showing an overall reduction in the activation of the PI3K/Akt pathway. Interestingly, the IM-resistant GIST-T1-Juke cells exhibited no decrease in P-C-KIT when treated with a high concentration of IM alone; however, PBOX-15 gave a strong decrease in P-C-KIT in these cells, which correlated with the effect seen on P-Akt. PBOX-15 appeared to overcome IM resistance. A negative regulator of the PI3K/Akt pathway, PTEN, was unaffected by either drug treatment, suggesting PTEN is not targeted. GIST-T1-Juke IM resistance is possibly not associated with loss of expression of PTEN as has been the case with other IM-sensitive GISTs [22].

KIT is regulated by the chaperone Heat Shock Protein 90 (HSP90) [23]. Recently Marino-Enriquez et al. [17] have shown CDC37, a co-chaperone of HSP90, to be a more specific target of KIT [17]. CDC37 associates with a large subset of HSP90 client proteins, primarily protein kinases [24] which are mainly involved in signal transduction, cell proliferation and survival. CDC37 interacts with KIT and regulates its expression, activation and downstream signalling targets in GIST [17]. In addition a positive feedback loop between casein kinase 2 (CKII) and CDC37 promotes the activity of multiple protein kinases and in turn CDC37 protects CKII from inactivation [25]. CKII is overexpressed in all cancers and is a key player in cell growth, survival and apoptosis, with over 300 substrates [26, 27, 28], its high expression in cancer cells is indicative of its importance in tumourigenesis.

We examined the expression of HSP90, CDC37, P-CDC37 and CKIIα (the active form of CKII [26]) in our cells after treatment. A down-regulation of HSP90 in IM-sensitive GIST cells was observed in response to IM alone, whereas in the IM-resistant GIST cells a decrease in HSP90 was elicited by PBOX-15. Both IM and PBOX-15 decreased P-CDC37 expression; however a stronger decrease was found with the combined treatment in both IM-sensitive and IM-resistant GIST cells, a similar effect to that observed with P-C-KIT. It is possible that in GIST-T1 PBOX-15 is independently inhibiting CDC37 function even though it does not appear to inhibit HSP90. In addition, PBOX compounds target microtubules and CDC37 regulates CKII, CKII binds to microtubules and has an important role in the maintenance of cell morphology [29]. PBOX compounds have been shown previously to depolymerize microtubules by inhibiting the assembly of purified tubulin, indicating the molecular target of PBOX is in fact tubulin [6]. Therefore, the PBOX disruption of the microtubules may prevent the binding of CKII to microtubules. To our knowledge this is the first time a MTA has been shown to inhibit CDC37 activity. A decrease in CDC37 possibly resulted in a downstream decrease in C-KIT, resulting from the loss of the regulatory feedback loop with CKII. This correlated well with a strongly reduced expression of CKIIα.

In conclusion, we have shown for the first time, the combined treatment of IM and PBOX-15 synergistically enhances apoptosis in both IM-sensitive and IM-resistant GIST cells, concomitant with a strong decrease in C-KIT, CDC37 and CKII, all of which play a significant role in maintaining the malignant phenotype of GIST. These findings indicate the potential of PBOX-15 to improve GIST treatment.

Notes

Acknowledgments

We would like to thank Novartis Pharma AG, Basel, Switzerland for their kind donation of imatinib. We would also like to thank Dr. Jonathan Fletcher for his initial advice with this project.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10637_2016_331_MOESM1_ESM.pptx (127 kb)
Supp Fig.1Imatinib/PBOX-15 gives a small amount of PARP cleavage in GIST-T1. Cells were treated with vehicle (Veh), imatinib (0.16 μM), PBOX-15 (0.6 μM) or combination for 24 h in GIST-T1 cells, cell lysates were prepared for western blot analysis. β-actin expression was measured as a loading control. Results are representative of three separate experiments. (PPTX 126 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Paula Kinsella
    • 1
  • Lisa M. Greene
    • 1
  • Sandra A. Bright
    • 1
  • Jade K. Pollock
    • 1
  • Stefania Butini
    • 2
  • Giuseppe Campiani
    • 2
  • Sebastian Bauer
    • 3
  • D. Clive Williams
    • 1
  • Daniela M. Zisterer
    • 1
  1. 1.School of Biochemistry and Immunology, Trinity Biomedical Sciences InstituteTrinity College DublinDublin 2Ireland
  2. 2.European Research Centre for Drug Discovery & Development, DBCFUniversity of SienaSienaItaly
  3. 3.West German Cancer Center, Medical OncologyEssenGermany

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